The chick auditory system is similar to that in mammals and has served as a valuable research tool in the study of sound processing, neural circuit development and degeneration for the past 40 years, mainly because of its accessibility throughout development. Until recently the tools to make genetic manipulations in the system, either gene overexpression or knockdown, have been limited mostly to early embryonic times. Recently transposon-based vectors have been shown to express in chick. The advantage of these vectors is that they integrate into the chick genome when introduced by in ovo electroporation at E2 and can be regulated in a temporal manner, meaning the genes are integrated at E2 but not expressed until the desired time. TrkB, the growth factor receptor that binds the neurotrophin BDNF, has an interesting and unique expression pattern in the two brainstem auditory nuclei, nucleus magnocellularis (NM) and nucleus laminaris (NL). NL is a laminar layer of bidentate neurons receiving excitatory input dorsally from ipsilateral NM axons, and ventrally from contralateral NM processes. From the start of synaptogenesis, TrkB expression increases in NL, segregates to the ventral dendrites, and remains there through hatching. In central neurons in mammals TrkB is crucial for dendrite growth, arborization and maintenance of synapses. Many of these studies have been done on neurons in culture, far fewer in vivo in transgenic animals, and without the fine temporal ability to regulate expression in specific cells. In addition, there are 2 major isoforms of TrkB and both are present in mammals and in the chick brainstem. We propose to exploit the use of transposon-based vectors in the chick to regulate TrkB isoform expression at specific times in development to dissect their roles in cell migration, dendritogenesis, synaptogenesis, and the refinement and maintenance of NM/NL synaptic connections. The Tol2-doxycycline regulated vectors reported by Sato et al. (2007) will be used with a TrkB-shRNA-mir30 expression cassette to regulate TrkB isoform knockdown. The techniques pioneered here, stable integration and temporal regulation of shRNA expression and TrkB gene expression at later embryonic stages in specific cells will be of great use others who study mid to late developmental processes in chick embryos. PUBLIC HEALTH RELEVANCE: The auditory circuit of the chick is similar to that in mammals and much more accessible to study, yet until recently the techniques to understand the molecular basis of development and degeneration of the system have been lacking in the chick. We will use a newly developed gene expression system to temporally regulate important molecules in specific cells in the chick auditory system to access their roles in the development and maintenance of proper neural connections